Immunization with Heat Shock Protein A and γ-Glutamyl Transpeptidase Induces Reduction on the Helicobacter pylori Colonization in Mice.

The human gastric pathogen Helicobacter pylori (H. pylori) is a successful colonizer of the stomach. H. pylori infection strongly correlates with the development and progression of chronic gastritis, peptic ulcer disease, and gastric malignances. Vaccination is a promising strategy for preventing H. pylori infection. In this study, we evaluated the candidate antigens heat shock protein A (HspA) and H. pylori γ-glutamyl transpeptidase (GGT) for their effectiveness in development of subunit vaccines against H. pylori infection. rHspA, rGGT, and rHspA-GGT, a fusion protein based on HspA and GGT, were constructed and separately expressed in Escherichia coli and purified. Mice were then immunized intranasally with these proteins, with or without adjuvant. Immunized mice exhibited reduced bacterial colonization in stomach. The highest reduction in bacterial colonization was seen in mice immunized with the fusion protein rHspA-GGT when paired with the mucosal adjuvant LTB. Protection against H. pylori colonization was mediated by a strong systemic and localized humoral immune response, as well as a balanced Th1/Th2 cytokine response. In addition, immunofluorescence microscopy confirmed that rHspA-GGT specific rabbit antibodies were able to directly bind H. pylori in vitro. These results suggest antibodies are essential to the protective immunity associated with rHspA-GGT immunization. In summary, our results suggest HspA and GGT are promising vaccine candidates for protection against H. pylori infection

Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.

Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain

Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.

Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain

<i>H</i>. <i>pylori</i> colonization in mouse stomachs after immunization.

<p>BALB/c mice (n = 10) were immunized intranasally on days 0, 14 and 21 with 30 μg antigen, with or without 10μg adjuvant, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130391#pone.0130391.t001" target="_blank">Table 1</a>. The same volume of PBS was used as a negative control. Three weeks after the final vaccination boost, mice were orally challenged four times with <i>H</i>. <i>pylori</i> B6. The level of gastric <i>H</i>. <i>pylori</i> colonization was determined by real-time quantitative PCR four weeks post challenge for each mouse. Data are expressed as mean ± S.D. Significant differences between indicated groups are presented as P values.</p

Indirect immunofluorescence confirmed that anti-serum collected from immunized mice directly binds <i>H</i>. <i>pylori in vitro</i>.

<p>No immunofluorescence was detected in the absence of antigen specific pcAb (A and B). Positive indirect immunofluorescence signals indicate binding of rHspA-GGT pcAb with <i>H</i>. <i>pylori</i> 26695 (C) and the existence of <i>H</i>. <i>pylori</i> (D). This study was performed twice, yielding similar results.</p

Vaccine formulations in each group.

<p>Vaccine formulations in each group.</p

Candidate antigens and immunization schedule used in this study.

<p>(A) Three His-tagged recombinant proteins were designed to be used as candidate antigens: full length HspA (rHspA), the catalytic domain of GGT (rGGT), and a fusion protein composed of rHspA and rGGT linked by two lysine residues (rHspA-GGT). (B) Recombinant proteins were purified by nickel ion affinity chromatography and gel-filtration chromatography. Recombinant proteins were then analyzed by SDS-PAGE. (C) The schedule for vaccine immunization, determination of cytokines secretion, and antigen-specific antibodies. (D) The schedule for vaccine immunization and analysis of <i>H</i>. <i>pylori</i> colonization.</p

Antigen specific antibody responses elicited by immunization.

<p>Two weeks after immunization, mice in each group (n = 5) were bled and the sera were collected for analysis of the specific IgG antibody and its subtype. (A) Specific IgG antibody titers, for antibodies raised against rHspA-GGT and <i>H</i>. <i>pylori</i> 26695 lysate, were measured by ELISA. (B) The levels of specific IgG1 and IgG2a, against rHspA-GGT, were determined from serum samples tested by ELISA. (C) Immunized mice were sacrificed 4 weeks after challenge and the supernatants of homogenized stomachs and intestines were collected for detection of specific sIgA levels, against rHspA-GGT. Data are presented as mean ± S.D (n = 5). (D) Regression analysis of bacterial loads and the level of rHspA-GGT specific serum IgG. **P < 0.01 compared with indicated groups, ***P < 0.001 compared with indicated groups, ns: not significant.</p

Immunization induced cytokine responses in murine spleen cells.

<p>Two weeks after the last booster, spleen cells of mice (n = 5) in each group were stimulated for 72 hours with antigen rHspA-GGT (5 μg/ml). The supernatants were harvested, and the cytokine levels of (A) IFN-γ, (B) IL-17A, (D) IL-4, and (D) IL-5 were determined by ELISA. Data are expressed as mean ± S.D. *P < 0.05 compared with all other groups or indicated groups, **P < 0.01 compared with indicated groups, ***P < 0.001 compared with indicated groups, ns: not significant.</p

Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering